Hypothesis: AN ECTOPIC SYNTHESIS OF THE MELANIN IN ADIPOSE MAY HELP TO COUNTERACT SECONDARY COMPLICATIONS OF OBESITY Manpreet Randhawa, Tom Huff, Julio.

Hypothesis: AN ECTOPIC SYNTHESIS OF THE MELANIN IN ADIPOSE

MAY HELP TO COUNTERACT SECONDARY COMPLICATIONS OF OBESITY

Manpreet Randhawa, Tom Huff, Julio C. Valencia, Zobair Younossi, Vikas Chandhoke, Vincent J. Hearing, Ancha Baranova

Why obesity is so dangerous ?

whyfiles.org/276metabolic_syndrome/

METABOLIC SYNDROME

Common complications :

Cardiovascular diseasesDiabetes type II

Non-alcoholic fatty liver disease (NAFLD)Non-alcoholic steatohepatitis (NASH)

Prevalence of the metabolic syndrome increases with age National Health and Nutrition Examination Survey III, 1988-1994

(8814 US adults)

Males

Females

Prevalence of metabolic syndrome increases with obesity

, blacks;    , Hispanics;    , whites.

U.S. INCIDENCE OF DIABETES (BOTH TYPES): CDC graph

Cost burden of obesity (U.S. only)

Direct cost only; 17% of total direct cost of heart disease, independent of stroke

Direct and indirect costs

Direct and indirect costs

Direct cost only; 17% of total direct cost of hypertension

Health-Care Costs for Obesity Top Those Related to Smoking

Obesity is associated with an average increase in hospital and outpatient spending of $395 a year

There has definitely been a feeling that smoking, drinking and

substance abuse are bigger problems

than obesity. This isn't the case.

Males

Females

Prevalence of metabolic syndrome increases with obesity

, blacks;    , Hispanics;    , whites.

Some obese people stay healthy

despite very high BMI(even within bariatric cohort)

WHY?

Glass half full or half empty?

Non-Alcoholic Liver Disease as a model

for the study of the secondary complications of obesity

From: Ariel E. Feldstein and Marsha H. Kay, ACG website

The majority of individuals with NAFLD have no symptoms and a normal examination

NAFLD affects up to 20 % of adults and nearly 5 % of children.

NASH 2-5 % of adult Americans; up to 20 % of obese subjects.

Can we treat NAFLD ? Not really. Just try to lose weight….

increased risk of hepatocellular carcinoma

Liver transplantation Fibrosis

For these who thinks that fat in the liver is microscopic change

IMPORTANT QUESTION: WHY SOME PATIENTS WITH VERY HIGH BMI (>45)

STAY FREE OF LIVER DISEASE?

ARE SOME PROTECTIVE FACTORS OR PREDISPOSING FACTORS INVOLVED?

ARE THESE FACTORS PRESENT IN THE LIVER OR IN THE FAT?

Results of the visceral adipose profiling

(40K chip)

Results of the Liver biopsy profiling (5K chip)

Dataset was downsized

> 2.0 in the liver (NASH vs OC)= pro-NASH genes in the liver

< 0.5 in the liver (NASH vs OC)= hepatoprotective genes in the liver

> 2.0 in the adipose (NASH vs OC)= pro-NASH genes in the fat

< 0.5 in the adipose (NASH vs OC)= hepatoprotective genes in the fat

Obese Patients with NASH

vsObese patients

without liver pathology

TNF-α and IL6 regulated genes expressed in adiposeplay a prominent role in the development of NASH

Genes that are regulated by TGF-β signal

in adipose are important for the development

of the primary phenotype

of the morbid obesity

Major finding: Adipose of the NASH patients

overexpresses a number of genes encoding secreted,

mostly pro-inflammatory molecules. IL18 interleukin 18 (interferon-gamma-inducing factor)

CCL26 chemokine (C-C motif) ligand 26

CCL20 chemokine (C-C motif) ligand 20

CTSB cathepsin B

B2M beta-2-microglobulin

UMOD uromodulin (uromucoid, Tamm-Horsfall glycoprotein)

PROC protein C (inactivator of coagul. factors Va and VIIIa)

FCN1 ficolin (collagen/fibrinogen domain containing) 1

CLU clusterin (complement lysis inhibitor, apolipoprotein J)

F3 coagulation factor III (thromboplastin, tissue factor)

Other cells embedded in adipose, including macrophages

Visfatin, Vaspin, Apelin, PAI-1, MCP1, other pro-inflammatory molecules

IL-6 IL-8

NAFLD and NASH develop under the influence

of the adipokines synthetized in the visceral adipose

ANOTHER study: Profiling of adiponectin, resistin,

visfatin, apelin, TNF-alpha, IL-6, IL-8, IL1, IL1R, sIL-6

in the serum of patients with and without NAFLD

100 samples collected and profiled;

Correlation analysis with seven clinical parameters is completed;

Visfatin TNF-α

IL-8 IL-6C. D.

Controls I(N=38)

Controls II(N=12)

NAFLD (N=45)

SS (N=19)

NASH (N=26)

Me

an

(p

g/m

l)

Controls I(N=38)

Controls II(N=12)

NAFLD (N=45)

SS (N=19)

NASH (N=26)

1

10

100

1000

A.

Me

an

(p

g/m

l)

100

B.M

ea

n (

pg

/ml)

Controls I(N=38)

Controls II(N=12)

NAFLD (N=45)

SS (N=19)

NASH (N=26)

100100

10

11

Controls I(N=38)

Controls II(N=12)

NAFLD (N=45)

SS (N=19)

NASH (N=26)

Me

an

(p

g/m

l)

10

1000

100

TNF-α

Controls I(N=38)

Controls II(N=12)

NAFLD (N=45)

SS (N=19)

NASH (N=26)

Me

an

(p

g/m

l)

10

1000

B.

100

TNF-α, pg/ml

Obese Healthy (N = 38)

1.91+/-0.25

Lean Healthy (N = 12)

2.3+/-0.39 < 0.0001

Lower than normal levels of TNF-αmay prevent the onset of NAFLD

in morbidly obese patients.

*

General Theme: Visceral fat in some obese subjects

remains inert; Something protects obese people with

inert fat from metabolic syndrome

Expression profiling in human OBESITY

• Comparisons of the visceral fat samples;

• >50 patients sampled during bariatric surgeries;

• 9 lean peoples donating their kidney;

Fat Tissue

Stratagene human Ref. RNA

Isolate total RNA

Reverse Transcribe tocDNA, label with Cy3

Reverse Transcribe to cDNA, label with Cy5

Mix, Hybridize and Scan

Linearly Amplify RNA Linearly Amplify RNA

METHOD :

Four comparisons were performed:

• Morbidly obese (N=50) vs. Controls

• Diabetic obese vs. normoglycemic obese

– No significant genes found

• Diabetic obese (N= 9) vs. Controls

• NormoGlycemic Obese (N=22) vs. Controls

DMO = Obese with diabetes; MO =Obese without diabetes;

NGO = NormoGlycemic Obese

All comparison were performed

vs. Non-Obese Controls

A LIST OF MELANOGENESIS RELATED GENES DIFFERENTIALLY

EXPRESSED IN OBESE ADIPOSE• Genes fold difference Accession number

TYRP1 2.66 AA668457

DCT 2.17 N27147

RAB27A 2.47 AI309109

RAB27B 2.6 R39044

MITF 1.5 N67822

SOX 1.36 AA976578

CHS1 1.22 N74383

Tyrosinase itself was not present on the chip

Expression of TYR, TYRP1 and DCT in lean and obese adipose samples

0

20

40

60

80

100

120

140

TYR DCT TYRP1

Re

lati

ve

ex

pre

ss

ion

un

its

Lean Adipose

Obese Adipose

Expression of TYR, TYRP1 and DCT in adipose tissue as revealed by

Quantitative RealTime-PCR

P < 0.007 P< 0.024 NS

Fontana-Masson stain of human adipose tissue demonstrates melanin pigment (black staining) mainly

in the periphery of the adipocytes.

Cryoslicing of visceral adipose

Melanin detection Fontana-Masson kit